Linearizer transcriptional circuits enable gradual gene expression in response to inducer molecule concentrations and can be used to enhance the yield and quality of mAbs, multispecifics, and rAAV products. This study compares the impact of random and targeted integration on the performance of two Linearizer circuits in CHO cells. Our results show that targeted integration delivers optimal linearizer performance (broad induction range and strong linear dose-response) by maintaining stoichiometric equivalence of circuit components. By optimizing linearizer circuit performance in CHO cells, we are paving the way towards unprecedented levels of real-time control and optimization of cell culture processes.

The critical step for clinical trials is having regulatory toxicology (RT) material, which depends on the availability of clones. Pfizer's site-specific integration system accelerates medicine development by reducing development times. This study demonstrates that material from non-clonal pools is comparable to clonal cell lines, with no significant trends in performance or differences in product quality. This strategy enables the acceleration of RT studies, thereby saving time without compromising quality.

Traditional cell line development relies on random transgene integration, and its intrinsic variability often requires more time and effort for clone selection and process development. This presentation will describe a site-specific integration approach to accelerate cell line development by targeting integration of transgenes to support high and stable antibody expression.

A new automated cell line technology platform designed to enhance standardization, reduce errors, and ensure stability in CHO cell line development for monoclonal antibodies. This high-throughput system accelerates timelines, delivers stable pools with yields of 3 g/L, and increases the likelihood of achieving monoclonal cell lines with titers of 4–6 g/L cost-effectively.

Significant progress has been made in the field of gene therapy through the development of AAV vectors that address multiple indications. However, there is a risk that these therapies may elicit inflammatory innate immune responses, which pose safety concerns and can diminish the overall effectiveness of gene therapy. Furthermore, rapid, robust and reliable in vitro methods to pre-emptively assess the immunogenicity of AAV vectors prior to patient administration are lacking. The development of such in vitro models and assays for screening gene therapy products early in the drug discovery process, could aid in the discovery of high-quality AAV gene therapy candidates with ameliorated immunogenicity. Using cell line engineering and flow cytometry approaches, we demonstrate the feasibility of developing multiple assays to measure the comparative innate immunogenicity of AAV vectors comprised of different capsids and genomes. Such efforts have the potential to improve product safety and shorten the development timeline of AAV gene therapy products from the lab to the clinic.

The literature has shown that Bak/Bax double-knockout mutants achieve higher titers during production, likely owing to an increased cell density from an impaired cell-death pathway. It has also been shown that Bak/Bax knockouts are not compatible with MTX selection. We sought to identify whether Bak/Bax knockouts were compatible with MSX-based selection and whether these mutants were able to achieve higher titers in our fed-batch production run.

As increasing numbers of monoclonal antibodies are developed to treat various diseases including cancer, autoimmune, and infectious diseases, there is a need to rapidly identify molecules easily expressed in cultured cells, particularly CHO cells. While AI has been extensively used to predict protein folding, its application in predicting manufacturability is limited. Using a novel AI approach, we developed a library of Vhh molecules and assessed their expression in CHO cells, identifying high and low expressors to train our AI tool. This approach will allow us to discover design rules to improve antibody expression, increasing the discovery of manufacturable molecules.

Developing high-yield, stable CHO cell lines is crucial for biotherapeutics, but traditional CLD platforms are slow and low throughput. We present a high-throughput, transposon-based CLD platform integrating chemical transfection, transposase, and CHO cells in 24-/96-well formats for batch and fed-batch production. Robust monitoring ensures optimal quality, accelerating stable pool generation and lead cell line selection while reducing CLD timelines. Successfully applied to monoclonal and complex bispecific antibodies, this system enhances productivity, quality, and efficiency, representing a significant advancement in biopharmaceutical development.

Chinese hamster ovary (CHO) cells are the main system for producing biopharmaceuticals, but they suffer from instability, affecting their long-term productivity. We created the first models for predicting long-term CHO cell stability due to changes in chromatin modification levels and methylation. Multilayer perceptrons are the best-performing models, reaching an F₁ score of 59.1% and a Matthews correlation coefficient of 19.4% on this task. Furthermore, Shapley values and interpretable models are used to investigate model coefficients, contributing biological insight to this problem and helping focus future data collection efforts. We freely provide the models trained in this work.

This study investigates the role of protein-protein interactions within Chinese Hamster Ovary (CHO) cells that experience toxic byproduct production, which reduces cell viability and protein synthesis. Utilizing crosslinking and advanced mass spectrometry, we explore interactomes to uncover the molecular mechanisms linking them to phenotypically distinct CHO clones. Our findings highlight potential pathways for enhancing CHO cell productivity and inform future CHO cell engineering strategies aimed at improving biologics production.

Codomax has developed the Epi-MAX platform, a systems-level, omics-driven, and tRNA-centric cell line engineering platform, to produce difficult-to-express proteins at scale with retention of biofidelity. The platform leverages an alternative genetic code in which a cell's tRNA pool is reprogrammed in response to cellular stresses, including those associated with biomanufacturing. Here, I will highlight recent efforts to engineer Pichia and CHO cell lines for the enhanced production of difficult-to-express biologics using mass spectrometry-based insights into tRNA reprogramming in response to recombinant protein production.

Chinese hamster ovary (CHO) cell culture faces challenges with cysteine and tyrosine solubility, stability, and toxicity. Excess or deficiency of these amino acids impacts growth and product quality. Additionally, CHO cells overconsume BCAAs, producing inhibitory byproducts. This work engineered a CHO line with BCAT1 knockout and introduced genes for cysteine and tyrosine prototrophy, enabling growth without supplementation and reducing BCAA needs—enhancing biologics production efficiency.

A limited understanding of cellular metabolism often restricts rational bioprocess optimization, leading to reliance on empirical methods such as design of experiments (DoE). Analytical methods including mass spectrometry-based metabolomics offers valuable insights into cellular status, highlighting potential metabolic bottlenecks for enhancing productivity and product quality. Method development on targeted metabolomics and machine learning (ML)-based absolute metabolite quantification, and their applications for media optimization and process understanding will be presented.

Evaluation of sequence variants by NGS has been incorporated into Cell-Line Development (CLD) processes for protein biologics and retroviral vectors.  As a clone-selection and quality-control step, an in-house Illumina MiSeq platform is used to eliminate clones that showed sequence variants in GOI.  In addition, NGS was also tested for potential adventitious-pathogens detection.

Intensified, continuous manufacturing processes have been developed to address challenges in rAAV production, improving scalability, yield, and efficiency. We present an upstream perfusion platform optimized for high cell density, continuous harvest, and feeding strategies, along with transient transfection optimization to enhance productivity. These innovations lay the foundation for overcoming current production limitations, ensuring consistent, high-quality rAAV for large-scale gene therapy manufacturing and advancing future therapeutic applications.

Perfusion and intensified cultures reduce costs, shorten production durations, and maximize recombinant productivity. Using NISTCHO, an open-access living standard, we achieved a 3x productivity increase with shorter culture durations through media screening, culture additives, optimized feeds, and seeding/bleeding strategies. Improved specific productivity via N-1 culture and feed strategies highlights their role in cell culture optimization for continuous and intensified processes.